VMC 221: Veterinary Immunology and Serology (1+1)

PROCEDURE

• Establish the working dilution of the antigen and conjugate by checker board titration.
• Dilute the antigen in coating buffer to its optimal dilution and add 100μl of diluted antigen in each well of a polystyrene microtitre plate.
• Incubate the plate over night at 4 ⁰ C in a humid chamber.
• Wash the plate thrice using washing buffer.
• Add 100 μl of the blocking buffer to each well and incubate at 37 ⁰ C for 30 minutes
• Wash the plate thrice using washing buffer.
• Make 2 fold dilutions (1:100, 1:200, 1:400 and so on) of the test and reference sera. Add 100 μl to each well. Include a negative control.
• Incubate the plate for 1 h at 37 ⁰ C in humid chamber.
• Wash the plate thrice using washing buffer
• Add 100 μl of the prepared conjugate to each well. Incubate at 37 ⁰ C for 1 h.
• Wash the plate thrice using washing buffer.
• Add 100 μl of the working substrate (OPD) to each well. Leave for 15 minutes for the reaction to take place in a dark chamber.
• Stop the reaction by adding 100ml of stopping solution to each well.
• Take the OD values (optical density) at 490 nm in an ELISA reader.

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